Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.
To study the intracellular apoptotic signaling pathways, we have established a cell-free system, in which DNA fragmentation of the isolated mouse liver nuclei was induced with lysates from the Fas-activated cells. We have found that the inactive nuclease present in the intact cell cytosol is activated by a caspase-3-like protease and the activated nuclease induces the nucleosomal DNA fragmentation. We attempted the purification of the inactive nuclease from bovine liver cytosol. The partially purified nuclease was activated by recombinant caspase-3, and to a lesser extent by caspase-6. The activated nuclease was able to digest plasmid DNA in addition to induce the DNA fragmentation of nuclei. DFF-45, which is a subunit of heterodimeric protein leading to DNA fragmentation upon its digestion by caspase-3, is found to inhibit the activity of the activated nuclease. These results suggest that the inactive nuclease in cytoplasm is converted to the active form by caspases, and the activated nuclease enters into nucleus to induce the DNA fragmentation. It is suggested that DFF-45 may function as an inhibitory factor of the caspase-sensitive nuclease in vivo.
Mitamura
S
S
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Ikawa
H
H
Mizuno
N
N
Kaziro
Y
Y
Itoh
H
H
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000235:genetics; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology*; D014162:Transfection / Q000235:genetics; D019014:fas Receptor / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D019014:fas Receptor; D003851:Deoxyribonucleases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases